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Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay

Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assa...

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Autores principales: Ishkaeva, Rezeda A., Zoughaib, Mohamed, Laikov, Alexander V., Angelova, Plamena R., Abdullin, Timur I.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8869332/
https://www.ncbi.nlm.nih.gov/pubmed/35204274
http://dx.doi.org/10.3390/antiox11020391
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author Ishkaeva, Rezeda A.
Zoughaib, Mohamed
Laikov, Alexander V.
Angelova, Plamena R.
Abdullin, Timur I.
author_facet Ishkaeva, Rezeda A.
Zoughaib, Mohamed
Laikov, Alexander V.
Angelova, Plamena R.
Abdullin, Timur I.
author_sort Ishkaeva, Rezeda A.
collection PubMed
description Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors.
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spelling pubmed-88693322022-02-25 Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay Ishkaeva, Rezeda A. Zoughaib, Mohamed Laikov, Alexander V. Angelova, Plamena R. Abdullin, Timur I. Antioxidants (Basel) Article Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors. MDPI 2022-02-15 /pmc/articles/PMC8869332/ /pubmed/35204274 http://dx.doi.org/10.3390/antiox11020391 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Ishkaeva, Rezeda A.
Zoughaib, Mohamed
Laikov, Alexander V.
Angelova, Plamena R.
Abdullin, Timur I.
Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay
title Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay
title_full Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay
title_fullStr Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay
title_full_unstemmed Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay
title_short Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay
title_sort probing cell redox state and glutathione-modulating factors using a monochlorobimane-based microplate assay
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8869332/
https://www.ncbi.nlm.nih.gov/pubmed/35204274
http://dx.doi.org/10.3390/antiox11020391
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